RESEARCH ARTICLE Dystrophinopathy Phenotypes and Modifying Factors in DMD Exon 45–55 Deletion Javier Poyatos-García, MS ,1,2 Pilar Martí, PhD ,1,2 Alessandro Liquori, PhD ,3,4 Nuria Muelas, MD, PhD ,1,2,5 Inmaculada Pitarch, MD, PhD ,5,6 Luis Martinez-Dolz, MD, PhD ,7,8 Benjamin Rodríguez, PhD,9,10 Lidia Gonzalez-Quereda, PhD ,9,10 Maria Dami a, PhD ,5,6 Elena Aller, PhD ,11 Marta Selva-Gimenez, MS ,1,2 Roger Vilchez, MS,1,2 Jordi Diaz-Manera, MD, PhD ,12,13,14 Jorge Alonso-Pérez, MD,12,13,14 José Eulalio Barcena, MD ,15 Amaia Jauregui, MD, PhD,15 amez, MD, PhD ,13,16 Jesus Angel Aladrén, MD,17 Ariadna Fern Josep G andez, MD,17 Marisol Montolio, PhD ,18,19 Inmaculada Azorin, PhD,1,2 David Hervas, PhD ,20 Ana Casasús, MS,1,2 Marisa Nieto, BS,1,2 Pia Gallano, PhD ,9,10 Teresa Sevilla, MD, PhD ,1,2,5,21 and Juan Jesus Vilchez, MD, PhD 1,2,5,21 Objective: Duchenne muscular dystrophy (DMD) exon 45–55 deletion (del45–55) has been postulated as a model that could treat up to 60% of DMD patients, but the associated clinical variability and complications require clarification. We aimed to understand the phenotypes and potential modifying factors of this dystrophinopathy subset. Methods: This cross-sectional, multicenter cohort study applied clinical and functional evaluation. Next generation sequencing was employed to identify intronic breakpoints and their impact on the Dp140 promotor, intronic long non- coding RNA, and regulatory splicing sequences. DMD modifiers (SPP1, LTBP4, ACTN3) and concomitant mutations were also assessed. Haplotypes were built using DMD single nucleotide polymorphisms. Dystrophin expression was View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.26461 Received Oct 22, 2021, and in revised form Jul 22, 2022. Accepted for publication Jul 22, 2022. Address correspondence to Dr Vilchez, Neuromuscular and Ataxias Research Group, Health Research Institute Hospital La Fe (IIS La Fe), Valencia, Spain and Neuromuscular Research Group, Neuromuscular Referral Center, La Fe Hospital, Health Research Institute La Fe, Av Fernando Abril Martorell, 106, CP:46026, Valencia, Spain. E-mail: juan.vilchez@uv.es From the 1Neuromuscular and Ataxias Research Group, Health Research Institute Hospital La Fe (IIS La Fe), Valencia, Spain; 2Centre for Biomedical Network Research on Rare Diseases (CIBERER); U763, CB06/05/0091, Valencia, Spain; 3Hematology Research Group, Health Research Institute Hospital La Fe (IIS La Fe), Valencia, Spain; 4Centre for Biomedical Network Research on Cancer (CIBERONC); CB16/12/00284, Madrid, Spain; 5Neuromuscular Referral Center, European Reference Network on Rare Neuromuscular Diseases (ERN EURO-NMD), Universitary and Polytechnic La Fe Hospital, Valencia, Spain; 6 Neuropediatric Department, Universitary and Polytechnic La Fe Hospital, Valencia, Spain; 7Cardiology Department, University and Polytechnic La Fe Hospital, IIS La Fe, Valencia, Spain; 8Centre for Biomedical Network Research on Cardiovascular Diseases (CIBERCV), Valencia, Spain; 9Genetics Department, IIB Sant Pau, Hospital of Sant Pau, Barcelona, Spain; 10Centre for Biomedical Network Research on Rare Diseases (CIBERER), U705, U745, CB06/07/0011, Barcelona, Spain; 11Genetics Unit, Universitary and Polytechnic La Fe Hospital, Valencia, Spain; 12 Neuromuscular Disorders Unit, Neurology Department, European Reference Network on Rare Neuromuscular Diseases (ERN EURO-NMD), Hospital of Sant Pau, Barcelona, Spain; 13Autonomous University of Barcelona, Barcelona, Spain; 14Centre for Biomedical Network Research on Rare Diseases (CIBERER), U762, CB06/05/0030, Barcelona, Spain; 15Neuromuscular Section, Neurology Service, Cruces University Hospital, Barakaldo, Spain; 16Neurology Department, European Reference Network on Rare Neuromuscular Diseases (ERN EURO-NMD), GMA Clinic, Barcelona, Spain; 17Calahorra Hospital Foundation, Calahorra, Spain; 18Duchenne Parent Project Spain, Madrid, Spain; 19Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, Barcelona, Spain; 20Department of Applied Statistics and Operations Research, and Quality, Polytechnic University of Valencia, Valencia, Spain; and 21Department of Medicine, University of Valencia, Valencia, Spain Additional supporting information can be found in the online version of this article. © 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association. 793 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. ANNALS of Neurology evaluated via immunostaining, Western blotting, reverse transcription polymerase chain reaction (PCR), and droplet digital PCR in 9 muscle biopsies. Results: The series comprised 57 subjects (23 index) expressing Becker phenotype (28%), isolated cardiopathy (19%), and asymptomatic features (53%). Cognitive impairment occurred in 90% of children. Patients were classified according to 10 distinct index-case breakpoints; 4 of them were recurrent due to founder events. A specific breakpoint (D5) was associated with severity, but no significant effect was appreciated due to the changes in intronic sequences. All biop- sies showed dystrophin expression of >67% and traces of alternative del45–57 transcript that were not deemed pathogenically relevant. Only the LTBP4 haplotype appeared associated the presence of cardiopathy among the explored extragenic factors. Interpretation: We confirmed that del45–55 segregates a high proportion of benign phenotypes, severe cases, and isolated cardiac and cognitive presentations. Although some influence of the intronic breakpoint position and the LTBP4 modifier may exist, the pathomechanisms responsible for the phenotypic variability remain largely unresolved. ANN NEUROL 2022;92:793–806 utations in the DMD gene that cause a severe deficiency M of the dystrophin protein are responsible for Duchenne muscular dystrophy (DMD), which evolves with muscle The breakpoint (BP) position of del45–55 in the flanking introns has been invoked as a potential determi- nant of phenotypic variability. On the one hand, it is degeneration and weakness starting in early childhood, loss of known that these introns contain regulatory elements, ambulation by the age of 13 years, and death around the such as the promoter of dystrophin isoform Dp140, which patient’s 30s. Partial dystrophin deficiency gives rise to Becker plays a role in brain development and whose deficiency muscular dystrophy (BMD), defined by the preservation of may entail a risk of cognitive impairment,21 and also hosts ambulation beyond 16 years and a broader spectrum of the age several long noncoding RNAs (lncRNAs) that regulate of onset and severity.1 Other reported dystrophin phenotypes DMD expression.22 On the other hand, changes in the include isolated cardiomyopathy and isolated hyperCKemia intronic architecture at the deletion junctions may alter occasionally associated with pseudometabolic manifestations. splicing signals that could promote aberrant splicing Brain involvement expressed as neurodevelopmental, cogni- events.23 To address these issues in the pre–next genera- tive, or behavioral abnormalities can also be present.2 tion sequencing (NGS) era represented a cyclopean task The clinical severity caused by a given DMD gene because of the large size of the introns involved combined mutation depends, to a large extent, on its impact on the with the rarity of this mutation. open reading frame.3 However, the location within the In this paper, we aimed to evaluate the different gene is also relevant for maintaining transcript stability and phenotypes associated with del45–55 by conducting a the conformational structure of the protein necessary for its multicenter study and analyzing the intronic BPs changes adequate assembly and interaction with other proteins.4 that could account for the clinical variability. Simulta- Additionally, genetic factors in trans, including variants of neously we explored concurrent gene trans-modifying genes related to the inflammatory response, regeneration, factors, including the SPP1, LTBP4, and ACTN3 DMD and fibrosis (SPP1, LTBP4, CD40, and TSHBS1) and to genetic modifiers. muscle endurance (ACTN3), can act as modulators.5 Among the diverse DMD mutations replicable for DMD therapy, the mega-deletion of exons 45 to Patients and Methods 55 (del45–55) may be able to correct up to 47 to 62% of Patients total DMD cases recorded in the mutation databases.6–8 A search for patients harboring del45–55 in the DMD This in-frame deletion in the dystrophin central domain gene was carried out via a cooperative study across Spanish has been observed in asymptomatic subjects and patients neuromuscular centers led by the Universitary and Poly- with more severe muscular involvement or presenting with technic La Fe Hospital in Valencia and Hospital of Sant significant cardiopathy.6,7,9–13 Currently, there is no expla- Pau in Barcelona. In addition, to inquire about the genetic nation for this clinical heterogeneity. Therefore, although and clinical department files, the patient registries held by rescue of functional dystrophin at the cellular and organis- the Carlos III Institute of the National Spanish Health mal levels has been achieved by mimicking this mutation Service (Center for Biomedical Network Research on Rare by means of multiexon skipping (MES) with cocktails of Diseases registry, CIBERER) and the one supported by antisense oligonucleotides (AOs)14–17 or applying genome- the Duchenne Parent Project Spain association were also editing technology,18,19 it is necessary to deepen the under- screened. Index patients were contacted by their respective lying pathogenic mechanisms to make this model a feasible neurologists or pediatric neurologists and were invited to alternative to current advanced therapies.20 participate in the study. Data from the hospital or primary 794 Volume 92, No. 5 Poyatos-García et al: DMD Δ45–55: Clinical Profile and Modifying Factors care files were obtained from the attending physician after (5, 6); IV, severe (≥7). Cardiac involvement was defined acquiring consent from the patient or the closest relative according to international guidelines,25 based on the pres- available, and the approval of the local health service ence of left ventricular ejection fraction < 55% or frac- supervisor was also obtained. The protocol of the study tional shortening < 25% or the existence of morphological was approved by the ethics committee of Hospital UiP La abnormalities in the ventricular walls evaluated via echo- Fe (2018/0200), and informed consent was obtained cardiography or clinical diagnosis by the attending cardiol- according to the Helsinki Declaration and signed by the ogist. Respiratory dysfunction was defined as forced vital patients and their respective parents or guardians. capacity (FVC) < 60% or ventilator use to treat restrictive pulmonary insufficiency. Clinical Aspects A protocol including relevant clinical features (demo- NGS Targeted Panel Design and Sequencing graphic data, family history, development milestones, age A 2.683Mbp custom panel was designed using at onset, age at symptom presentation, and age at signifi- SureDesign software (Agilent Technologies, Santa Clara, cant disease milestones), full neurologic and functional CA). Probes targeted all the DMD exons and the complete assessment using a modified Vignos scale (Supplementary 44- and 55-intron sequences. It also contained the SPP1, Table 1), and cardiologic and respiratory evaluations was LTBP4, and ACTN3 DMD modifiers and genes involved applied to every patient by their respective specialist at in myopathy.26 DNA libraries were prepared according to their treatment center. the SureSelectQXT Target Enrichment for Illumina Multi- For cognitive evaluation in adults, the protocol plexed Sequencing protocol and sequenced on a Miseq plat- included a checklist inquiring about neurodevelopmental form (Illumina, San Diego, CA) using the V3-150 cycles milestones, school and social performance, and behavioral cartridges. Files were obtained using the MiSeq Reporter and psychiatric disturbance. When any consistent abnor- 2.6.2.3 and Illumina RTA 1.18.54. Reads were mapped to mality was reported, a full assessment was recommended the human reference genome (GRCh37/hg19). Data analy- using the Wechsler Adult Intelligence Scale. A formal cog- sis was performed using Alissa and SureCall (Agilent nitive evaluation was executed systematically by the same Technologies). neuropsychologist on all pediatric patients. The study comprised a battery of validated tests employed in DMD BPs and lncRNA Identification evaluation24 covering different cognitive functions as fol- Primer pairs were designed to amplify 500bp of the dele- lows: general intelligence assessed with the Raven’s Col- tion junctions according to each BP position inferred from ored Progressive Matrices test, working memory evaluated NGS alignment in introns 44 and 55 and to detect the using the Digit Span subtest of Wechsler Intelligence lncRNAs nclINT44s, nclINT44s2, nclINT55s, and Scale for Children version IV, phonological processing nclINT55a.22 Polymerase chain reaction (PCR) was per- and verbal fluency evaluated using the respective Nepsy II formed using the PCR Master MIX (Thermo Fisher Sci- battery subtests, and executive functions and problem- entific, Waltham, MA), and the products were resolved solving capacity using the Tower of London test. Emo- on 1% agarose gel. BP junction amplicons were purified tional and behavioral problems were evaluated using the using ExoSAP-IT (Applied Biosystems, Waltham, MA) Child Behavior Checklist parent’s questionnaire. and analyzed via Sanger sequencing. A structured questionnaire inquiring about family data, main clinical features, and age at presentation of sig- Dystrophin Expression Analysis nificant milestones was administered via phone interview Available diagnostic muscle biopsies stored in liquid nitro- with patients or close relatives when the patient was not gen were used for dystrophin expression analysis, RNA able to attend a face-to-face visit. isolation, and reverse transcription PCR (RT-PCR). RNA Subjects harboring the deletion were classified into was extracted from muscle cryosections (RNeasy mini kit; one of the following phenotypes: (1) isolated hyper- QIAGEN, Hilden, Germany). RT was performed using CKemia, including plainly asymptomatic or oligo-/ 750ng of total RNA with SuperScript IV Reverse Tran- paucisymptomatic when associated with minor symptoms scriptase (Invitrogen, Waltham, MA), and nested PCR of such as pseudometabolic symptoms (myalgia and cramps cDNA samples was carried out using specific primer pairs or rhabdomyolysis induced by exercise or strenuous fac- (hybridizing in DMD exons 41, 43, 44–58, 59, and 60) tors); (2) BMD phenotype; and (3) isolated cardiopathy. as previously described.27 PCR products were analyzed on Myopathic severity impairment was classified into 4 grades 1% agarose gels, and DNA was purified (Gel Extraction according to the modified Vignos scale as follows: I, Kit; Omega Bio-Tek, Norcross, GA) and validated via asymptomatic (grade 0–1); II, mild (2–4); III, mild–severe Sanger sequencing. November 2022 795 ANNALS of Neurology Duplex droplet digital PCR (ddPCR) was performed best practices from the Broad Institute. Single nucleotide with 4μl of cDNA, using the QX200 Droplet Digital polymorphism (SNP) genotypes were extracted from all PCR system (Bio-Rad Laboratories, Hercules, CA). Two positions in the DMD-sequenced region. Variants were specific ddPCR custom assays were used in each reaction, annotated using Genome Aggregation Database allele fre- with probes designed to hybridize the DMD 44–56 exon quency (European non-Finnish population). Only variants junction (FAM-labeled) and the 44–58 junction with allele frequencies of ≥0.3 and <0.5 were kept for fur- (HEX-labeled). Data were analyzed with QuantaSoft ther analysis. For haplotype construction of each group, Analysis Pro Software (Bio-Rad Laboratories). The con- the SNP (v) and reference (r) forms of each subject were centration (copies/μl) of both transcripts was used to considered. A plot representing only the predominant determine the percentage of the del45–57 transcript in form (r/v) in each deletion group was plotted. each sample as follows: [(copies/ul 44–58)/(copies/ul 44– 58 + copies/ul 44–56)]*100 .28 DMD Modifiers For muscle immunostaining, 7μm cryosections were Within the known DMD genetic modifiers, we selected coincubated with the primary monoclonal antibodies to explore SPP1, LTBP4, and ACTN3 for relevance and antidystrophin (NCL-Dys2; Novocastra Laboratories, logistic reasons. For that purpose, the NGS gene panel Newcastle Upon Tyne, UK; 1:50), and rat monoclonal was used in the index cases and ad hoc Sanger sequencing anti–laminin-2 (Sigma-Aldrich, Burlington, MA; 1:500). in the secondary cases. The criteria applied were as fol- The appropriate secondary antibodies were used (Alexa low5: the presence of G risky allele in SPP1 (dominant Fluor 594 goat antimouse and Alexa Fluor 488 goat model), the LTBP4 IAAM/IAAM beneficial haplotype, antirat; 1:200, Invitrogen). Sixteen-bit images were and ACTN3; in Model 1, we included the TT and TC as acquired using an LSM800 confocal microscope (Zeiss, harmful alleles, whereas in Model 2, the heterozygous Oberkochen, Germany) at 200 magnification. allele was taken as an intermediate. Presence of concurrent Dystrophin detection via Western blotting from the mutations in the other genes within the working panel skeletal muscle biopsies was performed as previously were also analyzed (Supplementary Table 2). described,29 using the following primary antibodies: anti- dystrophin (NCL-Dys1, 1:40, Novocastra) and anti–α- Statistical Analysis actinin (A7732, 1:3,000, Sigma-Aldrich). Horseradish Nonparametric tests were used to compare continuous peroxidase–conjugated antimouse IgG (ab6808, 1:2,000, variables within the different groups (p < 0.005). A Bayes- Abcam, Cambridge, UK) was used as the secondary anti- ian ordinal regression model was applied to assess the body. Membranes were visualized using chemilumines- effect of deletion location, lncRNA preservation profile cence (ECL PLUS, Thermo Fisher Scientific) and groups, and DMD-modifying factors on the severity of Amershand Imager 600 (GE Healthcare, Chicago, IL). skeletal muscular involvement. Bayesian logistic regression Dystrophin intensity was normalized to α-actinin using was used to evaluate the effect of dichotomous variables the ImageJ software. (cardiac involvement). Cognitive involvement was only exploratory due to the difference in diagnostic methodol- Deletion Junction Characterization ogy between adult and pediatric populations. The criteria In the intronic sequences near BPs, repetitive elements applied are listed in Table. All statistical analyses were per- were searched using the RepeatMasker program (http:// formed using R (v3.5.3), R package brms (v2.8.0), and repeatmasker.org/cgi-bin/WEBRepeatMasker) and inverted clickR (v0.4.32). repeats were obtained using the Palindrome program (http:// emboss.bioinformatics.nl/cgi-bin/emboss/palindrome). The Results Human Splicing Finder V3 tool was used to analyze the Patient Clinical Features splicing regulatory elements (SREs) and the potential Full clinical evaluation was carried out on 39 subjects, pseudoexon activation at the deletion junctions, as previously including 23 index and 16 secondary cases. Reliable data described.30 For these analyses, we searched for SREs and from 18 additional subjects, either deceased (n = 13) or splice sites in patient sequences, including rare variants, unable to attend a face-to-face visit (n = 5), were obtained rather than reference ones. via telephone interviews and from clinical records. Supple- mentary Table 1 summarizes the clinical data of the Founder Effect Haplotype Analysis 57 patients in this series and specifies the assigned deletion Haplotype analysis was performed for the groups of index group according to the respective index case. The analysis patients who shared the same intronic BPs. Variants were of the 23 pedigrees demonstrated X-linked transmission in called using the GATK HaplotypeCaller tool31 following all but 5 of the kindred. 796 Volume 92, No. 5 Poyatos-García et al: DMD Δ45–55: Clinical Profile and Modifying Factors TABLE. Bayesian Regression Analyses Evaluating the Effect of Deletion Location, lncRNA Pattern, and Modifying Factors on Muscle Severity Impairment Grade and Cardiac Involvement Muscle Severity Impairmenta Cardiopathyb Deletion, n = 55 D1, n = 25 Ref. Ref. D2, n = 5 0.525 (0.067–3.817) 0.396 (0.028–4.161) D3, n = 6 2.098 (0.257–15.275) 2.915 (0.39–22.022) D4 (Dp140 ) , n = 6 1.957 (0.02–215.243) 0.876 (0.009–82.252) D5, n = 3 10.824 (1.314–98.235) 44.302 (2.075–2,530.511)c c FIGURE 1: Graphical representation of age at evaluation of D7, n = 4 0.196 (0.002–14.81) 4.656 (0.121–190.056) del45–55 cohort. Boxplot shows the median and range of D9, n = 3 0.33 (0.023–4.046) 0.047 (0.001–1.358) assessment age of the 3 diagnostic categories. There is a significant difference between the asymptomatic/ D10, n = 3 0.09 (0.001–3.271) 0.085 (0.001–3.62) paucisymptomatic group (median = 35.5, range = 8–87) and lncRNA, n = 55 the Becker muscular dystrophy (BMD; median = 65.5, range = 40–78) and isolated cardiopathy (median = 66, PPPP, n = 4 Ref. Ref. range = 32–88) groups (** p < 0.01, *** p < 0.001 according PPNN, n = 45 9.835 (0.217–593.61) 2.112 (0.062–79.874) to Kruskal-Wallis test). NNPP, n = 6 2.033 (0.024–206.376) 0.807 (0.009–68.4) Modifiers, n = 33 Figure 1 represents the age of patients at evaluation. SPP1 1.125 (0.135–8.59) 1.213 (0.132–10.35) The patients in the isolated hyperCKemia group were sig- LTBP4 0.576 (0.065–6.127) 40.829 (1.996–1,707.37)c nificantly younger than those in the BMD and isolated ACTN3 Model 1 0.639 (0.109–3.733) 0.486 (0.065–3.327) cardiopathy groups. The median age at onset in index patients was 12 years (range = 2–40 years). Muscle ACTN3 Model 2 0.519 (0.151–1.53) 0.733 (0.223–2.177) impairment in patients with BMD always occurred after c Age 1.086 (1.029–1.155) 1.072 (1.013–1.145)c the age of 20 years, but on most occasions (10/16) after Deletion 1 was used as a reference, in addition to the group con- the age of 40 years (late onset BMD). Approximately 44% taining the 4 lncRNAs. Age was considered as a cofounding variable (7/16) of patients with BMD lost ambulation, usually for all models. Deletions and lncRNA pattern groups containing only after 50 years of age. one subject were excluded from the analysis (D6 and D8). Evidence Cardiopathy debut was always after the age of for an effect was considered relevant when the 95% CI (confidence interval) did not include 1. The odds ratios and CIs are indicated for 20 years, with an increasing age-related incidence rate each comparison. In the lncRNA pattern, N = absence and (Fig 2A). Details of the available cardiological data are P = presence. listed in Supplementary Table 3. Four cases presented a Bayesian ordinal regression model. atrioventricular block requiring emergency pacemaker b Bayesian logistic regression model. c implantation, one of them at age 29 years, as an inaugural Statistically significant. symptom. Two others received an implantable cardioverter–defibrillator. In many instances, patients remained stable with medical therapy, but the cardiac Patients were divided into the following categories: transplant was indicated in 3 cases; one survived 9 years isolated hyperCKemia (53% of cases), further subdivided after the intervention, a second one died while on the into asymptomatic (n = 23) and paucisymptomatic waiting list, and the third one is still expecting the (n = 7). The BMD phenotype accounted for 28% of intervention. cases (n = 16); isolated cardiopathy occurred in 19% of None of the patients required respiratory attention, cases (n = 11). Cardiopathy was also present in 7 patients and routine evaluation tests always showed an FVC with the BMD phenotype. Cognitive and/or behavioral of >50%. disturbances were detected in 10 subjects, 9 of pediatric The prevalence of symptoms across all ages of the age, and all manifested hyperCKemia. The adult patient whole series is shown in Figure 2A. Whereas the propor- also presented cardiopathy. tion of asymptomatic patients dropped from 100% at age November 2022 797 ANNALS of Neurology Supplementary Table 4 collects the results of the cog- nitive assessment. Nine of the 10 pediatric patients evaluated presented abnormalities, including neurodevelopmental fea- tures (80%), reduced intelligence quotient (IQ) score (40%), speech abnormalities at phonological (88%) or verbal processing (50%), executive function (55%), and emotional– behavioral features (80% internalization and 70% externali- zation). The adult patient profile showed a similar profile. When the data were collected, 14 patients had died; cardiopathy was imputed as the causative factor in 6 sub- jects, 1 case presented as sudden death, and other medical or nonspecific causes were reported in the remaining 6 cases. The median age at death was 70 years (range = 52–88 years). Life expectancy in the Spanish male population has ranged from 76 to 79 over the past 20 years. Intronic BP Analysis Ten different deletions with specific intronic BPs were rec- ognized in the 23 index cases (Fig 3). Four of them were shared by several index cases (deletion groups D1–D4), FIGURE 2: Prevalence of clinical manifestations across age of whereas the other 6 (D5–D10) appeared in a single case. del45–55 series. (A) Global series (n = 57) prevalence of Within the shared deletion, D1 was present in 9 index symptoms at 4 age periods. (B) Same representation in the Deletion 1 group (n = 25). cases, D2 and D3 in 3 cases each, and D4 in 2 others. The genomic location of each BP and the deletion length of all 10 specific groups are presented in Supple- mentary Table 5. D4 and D8 disrupted the promoter 20 years to 22% after the age of 60 years, that of patients region of the dystrophin isoform Dp140 located in intron with symptomatic BMD rose to 52% in their 60s. The 44 (see Fig 3). In addition, Figure 3 and Supplementary prevalence of cardiopathy reached 43.5% at the age of Table 2 show the effects of the different deletions across 60 years (26% of them isolated). The proportion of symp- intronic lncRNAs sequences. Only D7 preserved all toms in the largest BP subgroup (D1) followed a similar lncRNA sequences, whereas most deletions partially trend to the whole series (see Fig 2B). disrupted these lncRNAs, leading to different preservation FIGURE 3: Schematic representation of the 10 specific deletion breakpoints of the del45–55 cohort. In the upper part, there is a schematic representation of the normal genomic structure spanning the del45–55 region. The promoter and first exons of the Dp140 and Dp116 isoforms are shown. The positions of the long noncoding RNAs (lncRNAs) 44s, 44s2, 55s, and 55as and their transcription directions are also represented. Each specific deletion is represented according to its intronic breakpoint position and labeled as D1 to D10. All deletions, except D4 and D7, disrupted the sequence of the lncRNA 55as containing the polyadenylation site, as indicated by asterisks. Dotted lines indicate the deleted segment. 798 Volume 92, No. 5 Poyatos-García et al: DMD Δ45–55: Clinical Profile and Modifying Factors (Figure legend continues on next page.) November 2022 799 ANNALS of Neurology profiles. D8 was the only one that deleted all the lncRNA contained 2 different subgroups of haplotypes, indicating sequences. the presence of 2 different founder events in the same spe- Deletion junction characterization revealed the pres- cific deletion (see Fig 6B). Globally, we identified 5 dis- ence of sequences capable of inducing DNA double-strand tinct founder mutations. break (DSB) and other features, such as insertions and microhomologies. Repetitive elements were found near BP Effect on Clinical Severity the BPs, with absence of extensive homology. Details are Table shows the result of the analysis of the diverse vari- given in Supplementary Table 5.32,33 The analysis of ables on clinical severity. Only a significant effect of the pseudoexon activation depicted the potential formation of D5 BP appeared on muscle severity impairment (odds ratio cryptic exons in all the deletion junctions (Supplementary [OR] = 10.824, 95% confidence interval [CI] = 1.314– Table 6), whereas the analysis of the SREs showed that 98.235) and the presence of cardiopathy (OR = 44.302, the deletions did not modify the exonic splicing enhancer/ 95% CI = 2.075–2,530.511). silencer ratio, which was close to 1 in all 10 (Supplemen- tary Table 7). Effect of the Intronic Sequences As represented in Table, no significant effect was observed Dystrophin Expression from the lncRNA profiles (presence, absence, or partial Dystrophin immunostaining in 9 available muscle biop- disruption) and the Dp140 disruption (deletions D4 and sies (from P1; P2; P4,2; P7; P8,1; P9; P17; P18; P22,1) D8) over the severity of skeletal muscle involvement and showed a correct dystrophin sarcolemma expression, with development of cardiopathy. The connection between a slight intensity reduction in some cases (Fig 4A). cognitive and behavioral impairment with Dp140 is Quantification of dystrophin expression via Western blot- depicted in Supplementary Table 4, where 8 of 10 cases ting showed high levels in all muscle samples, ranging showing cognitive abnormalities held a preserved pro- from 67 to 106% of a healthy control sample (see motor (Dp140+), whereas in the 2 others affected the Fig 4B). promotor was absent (Dp140 ). The RT-PCR analysis did not confirm the presence of pseudoexons (Supplementary Table 6). However, the Trans-Modifying Factors sample from P18 (D5 group presenting a severe pheno- No statistically significant effect of the risky SNPs in type) showed a significant number of the replicates with SPP1, LTBP4, or ACTN3 was found on the clinical sever- an additional skipping of exons 56 and 57, which led to ity of myopathy in the assessed patients (n = 33). How- transcripts with an in-frame deletion of exons 45 to ever, the absence of the LTBP4 IAAM/IAAM haplotype 57 (del45–57; Fig 5A). A subsequent specific amplifica- was associated with the presence of cardiopathy tion of this transcript (using a primer targeting the 44–58 (OR = 40.829, 95% CI = 1.996–1,707.37; Table). On exon junction) revealed its presence in all the samples. the other hand, concurrent heterozygous mutations were The quantification of the del45–57 transcript using present in 4 subjects (Supplementary Table 2), but no duplex ddPCR showed a residual expression, accounting relationship with the symptoms was observed (ie, meta- for <0.1% in all cases (see Fig 5B, C). bolic gene/pseudometabolic features). Haplotype Analysis Discussion Given that most of the subjects sharing the same deletions In this study, we analyzed a large cohort of patients with (D1–D4) were residing in the same geographical region dystrophinopathy, harboring the deletion of DMD exons (Supplementary Table 1), we performed a haplotype anal- 45 to 55, wherein data were collected across different neu- ysis to search for founder effects. All individuals within romuscular centers and evaluated using a systematic clini- their respective D2, D3, and D4 groups harbored the cal protocol. This mutation is highly infrequent, same haplotype (Fig 6A). However, the D1 prototype representing <2% of the cases recorded in DMD FIGURE 4: Dystrophin protein analysis of the 9 del45–55 muscle biopsies; healthy (C) and Duchenne muscular dystrophy (DMD) samples. (A) Representative images of dystrophin immunostaining in transverse sections revealing a proper sarcolemma protein location with a slight or mild staining reduction in some patients (DYS2, upper image). Laminin immunostaining (lower image) was used to localize muscle sarcolemma. Scale bar = 50μm. (B) Dystrophin quantification by Western blotting of total protein extract. Note the del45–55 dystrophin with a lower molecular weight than control, and absent protein in the DMD sample. Dystrophin levels were normalized to α-actinin signal and are represented as percent of healthy control. Data represent the mean of 3 technical replicates. Bars of the same shade indicate subjects from the same deletion group (D1 group = P1; P2; P4,2; P7; P8,1; P9. D4 group = P17. D5 group = P18. D9 group = P22,1). 800 Volume 92, No. 5 Poyatos-García et al: DMD Δ45–55: Clinical Profile and Modifying Factors FIGURE 5: Dystrophin expression analysis of 9 muscle cDNA samples. (A) Presence of an alternative transcript in P18 (D5) muscle cDNA. Nested reverse transcription polymerase chain reaction products from P18 (D5) and P1 (D1) showed an upper band that corresponds to the expected del45–55 transcript. In addition, P18 showed a lower band, revealing an additional skipping of exons 56 and 57. The intermediate band in P18, marked with an asterisk, was considered a heteroduplex formation. The identities of all bands were confirmed by Sanger sequencing. (B) One-dimensional fluorescence amplitude plots of the 2 ddPCR assays, quantifying the levels of the del45–55 transcript (upper part, FAM-labeled probe targeting the exon 44–56 junction) and the del45–57 transcript (bottom part, HEX-labeled probe targeting the exon 44–58 junction) in the nine samples. The horizontal lines indicate the threshold level established to discriminate the positive from the negative droplets (above and below, respectively). Three technical replicates per sample were run, and a no template control (NTC) was included as negative control. FAM= 6-carboxyfluorescein fluorophore; HEX= 6-carboxy-2’,4,4’,5’,7,7’-hexaclorofluorescein fluorophore. (C) Low percentage of the del45–57 transcript in all samples, determined as [(copies/ul 44–58)/(copies/ul 44–58 + copies/ul 44–56)]*100. Percentages are shown as mean (n = 3). Bars of the same shade indicate subjects from the same deletion group (D1 group = P1; P2; P4,2; P7; P8,1; P9. D4 group = P17. D5 group = P18. D9 group = P22,1). [Color figure can be viewed at www.annalsofneurology.org] registries.7 To the best of our knowledge, no previous Echigoya et al17 collected a total of 48 cases. As shown in studies have provided a series of significant sample sizes Figure 7, our data differ to some extent from Echigoya’s enabling a rigorous clinical and genetic analysis. Data review in the proportion of phenotype incidence, particu- available on this condition are based on the reporting of larly a higher rate of cardiopathy and a considerable pro- small case series or anecdotic descriptions from which portion of cognitive impairment. November 2022 801 ANNALS of Neurology FIGURE 6: Haplotype analysis of subjects with the shared deletions D1 to D4. (A) Plot representing a selection of single nucleotide polymorphism (SNP) forms within the DMD sequenced region from subjects of the deletion groups D1 to D4. For each SNP, both reference (r) and variant (v) sequences are considered, and only the predominant form is plotted, as gray (r) or red dots (v) according to its proportion within the subjects of each group (the position in the y-axis represents the proportion). Note that for all subjects from each D2 to D4 group, mostly a common SNP profile is depicted, except subjects from the D1 group showing 2 profiles. The position of the deletion breakpoints is represented as vertical dashed bars. The plot at the bottom represents SNPs reaching the selection criteria (allele frequency ≥ 0.3 < 0.5) along the entire DMD gene. (B) Magnification of the region of interest in the D1 group (chrX: 32,056,814–32,219,490), highlighting the 2 SNP profiles. The SNPs (r or v) common in the 9 subjects are represented in gray, whereas the subgroups of 6 and 3 subjects with distinct haplotypes are represented with green and red dots, respectively. The position of the dots along the y-axis is arbitrary. [Color figure can be viewed at www.annalsofneurology.org] There are several clinical aspects to be highlighted in to 40% of patients with BMD suffered a loss of ambula- our series, which allowed us to draw up a natural history tion or reached a high degree of motor disability, whereas profile of the condition. As a rule, hyperCKemia and a significant number of cases evolved with severe cardiopa- pseudometabolic features may be present since early child- thy demanding cardiac transplant or suffered from unex- hood and can continue to be the only manifestations in a pected serious acute heart events and sudden death, sizable number of cases even after the age of 60 years. Sec- indicative of threatening cardiac arrhythmias. Further- ond, although the incidence of cardiopathy falls in the more, data from affected deaths indicate a shorter life average of BMD reported series,34,35 this mutation tends expectancy. Finally, learning and cognitive features are to present a high propensity to early cardiac impairment other remarkable issues scarcely evaluated in this deletion. and isolated cardiopathy appearance. Third, this condition Signs of brain impairment were recorded in 23% of the does not appear to be as benign as supposed, because up total series; however, the proportion rose to 90% in the 802 Volume 92, No. 5 Poyatos-García et al: DMD Δ45–55: Clinical Profile and Modifying Factors groups and grades of severity and all cases showed high dystrophin expression, not accounting for the variable phenotypic severity. A central objective of our study was to characterize intronic BPs and establish their implications in the phenotypic variability. For this purpose, we devised an NGS- based targeted design complemented with Sanger sequencing for BP identification, which proved to be accurate and highly resolute compared to the classical approaches.32,41 Third- generation long-read sequencing has also enabled the accurate FIGURE 7: Comparison of the del45–55 clinical profile from identification of intronic BPs along DMD intron 44.33 The the Echigoya et al17 review series and our cohort. Note a higher proportion of asymptomatic/paucisymptomatic and analysis of the deletion junctions revealed the presence of cardiomyopathy cases (*19% isolated and 13% associated diverse repetitive elements in the vicinity of the deletion BPs with Becker muscular dystrophy [BMD]) in our series. that may contribute to the genomic instability, but no exten- Cognitive impairment was always associated with hyperCKemia,** which was absent in Echigoya’s work. The sive homology was found, eliminating the homologous unclassified group of Echigoya’s series correspond to recombination as the underlying mechanism. Nevertheless, 5 subjects with limited clinical information. we found a variety of elements associated with DSB forma- tion in all the deletion junctions, indicating that del45–55 does not arise from a single mechanism but from the involve- pediatric population, possibly because this group was sub- ment of a variety of factors that induce DNA instability and jected to a more rigorous cognitive evaluation. This figure DSB formation, as well as the involvement of different geno- is the highest rate of those reported in the DMD and mic repair pathways (Supplementary Table 5).32,33 BMD series,24,36 and it is worth mentioning that it has In keeping with another study that analyzed a rarely been reported as an isolated manifestation.2 del45–55 series, we did not find hotspots predisposing to The cognitive profile depicted by most of the evalu- BP recurrence.42 ated del45–55 patients was on par with the signature The haplotype study in our series revealed that all reported in DMD studies.37 The high rate of neu- index case groups harboring the same BP originated from rodevelopmental and behavioral features observed was also distinct founder mutations, except for the D1 group, in consonance with the reported BMD series.36,38 How- which turned out to be the result of 2 independent founder ever, these data are discrepant with a study observing a mutation events. The high rate of founder effects observed distinct BMD profile with preservation of full scale IQ in our series contrasts with their rare occurrence in the and higher impairment of performance scale over verbal BMD disease population, as only 2 similar founder effects scale. Differences in the battery of tests cannot explain the occur have been reported.43,44 In any case, this phenome- discrepancy, but differences in patient samples might; a non is indicative of the preservation of fertility. On the proportion of patients reported in Young et al study were other hand, this observation provided us with the chance not genetically characterized.38 to dispose of groups with sufficient size to enable the com- So far, several studies of BMD patient series have parison of the effect of BP locations on the outcomes. analyzed the clinical severity of different in-frame deletions In our analysis, we found a significant influence of in the dystrophin rod domain spanning exons 44 to 55. BP only in the D5 group with severe manifestations. In the Deletions presenting the most favorable phenotypes, such first instance, the RNA analysis of a subject from this as del45–55 and those that are exon-51 skippable, exhibit group revealed an alternative skipping of exons 56 and a dystrophin hybrid repeat conformation, preserving the 57, which might be induced by the changes in the splicing filamentous protein structure. As opposed to this, there sequences of this specific BP. However, later, the skipped are other deletions (eg, del45–47 and del 45–49) causing transcript was found in all biopsies, raising concerns about fractional repeats associated with greater severity.4,6,34,35,39 its pathogenicity. Alternative exon skipping events have Moreover, studies of internal variability and potential been detected in different pathological conditions45 and modifying factors in a single BMD mutation have scarcely are infrequent in healthy subjects.46 Furthermore, its resid- been addressed, such as the analysis of the impact of neu- ual expression in all the samples suggests that is presumably ronal nitric oxide synthase mislocalization in del45–55 a subproduct derived from multiexon-skipping events trig- muscle12 and the dystrophin amount in del45–47.40 gered by post-transcriptional introns.47 Otherwise, no evi- Regarding this issue, although the number of samples in dence of pseudoexon activation was found in any biopsies our study was limited, they were representative of distinct despite the positive in silico predictions. November 2022 803 ANNALS of Neurology We also performed an approximation of the disrup- (2018/0200). A.L. is a recipient of the GVA fellowship tion of the lncRNAs based on the BP location, and no APOSTD2021/212. N.M. and J.J.V. are funded by clinical impact was observed regarding the different preser- PI16/00316 supported by Instituto de Salud Carlos III vation patterns. Gargaun et al42 reported the specific (ISCIII) Madrid and by PROMETEO/2019075 PNNN profile (it preserves only the lncRNA44s) as pro- supported by Conselleria de Sanitat, Generalitat Vale- tective, but we could not confirm this, because it was pre- nciana. I.P. and M.D. are funded by Duchenne Parent sent in only one of our subjects (P19, D6). Nevertheless, Project Spain (2018/0541). P.G. and L.G.-Q. are funded these speculations should be rigorously addressed at the by PI18/01585 supported by ISCIII. T.S. is funded by RNA level to evaluate the role of lncRNAs in dystrophin PROMETEO/2018/135 (Conselleria de Sanitat, Gen- transcription.22 Similarly, we evaluated the effect of the eralitat Valenciana) and by PI19/01178 (ISCIII). BP location on the Dp140 promotor on the cognitive sta- We thank all the patients and family members for tus in the properly evaluated patients, not observing a their cooperation. We acknowledge the Data Science Unit straightforward relationship with its preservation. How- of IIS La Fe, Valencia for statistical analysis and the Geno- ever, this interpretation may be controversial, because the mic and Translational Genetics Unit of CIPF, Valencia Dp140 translation should be altered by the deletion of the for sequencing support. We are grateful to the collabora- translation start site at exon 51. However, Bardoni et al48 tion of “Càtedra de Malalties rares,” University of Barce- proposed the existence of alternative translation sites lona. We also thank Editage (Cactus Communications) located between exon 52 and exon 56, although this sug- for editing support. gestion has not been experimentally substantiated. The validated DMD genetic modifiers, including Author Contributions the 3 investigated in this study, have not yet been assessed J.P.-G., P.M., and J.J.V. contributed to conception and in BMD. Our exploratory analysis is limited by the small design of the study. All authors contributed to acquisition sample size (n = 33) that underpowers the statistical anal- and analysis of data. J.P-G., P.M., A.L., B.R., and ysis. Nevertheless, we found an effect between the LTBP4 J.J.V. contributed to drafting a significant portion of the and the presence of cardiopathy, which is an interesting manuscript or figures. observation that needed to be confirmed. Similarly, although heterozygous concomitant mutations were Potential Conflicts of Interest observed in several subjects that might have acted as dou- J.P.-G. has received a PhD grant from Fundacion Isabel ble trouble,49 we did not find any aggravating or confus- Gemio. J.J.V. has received grants from Fundacion Isabel ing manifestation over the phenotype in those cases. Gemio and ISCIII and has been a consultant to Bio- Overall, our study confirms del45–55 as a model Marin, Genzyme Therapeutics, and PTC Therapeutics. capable of segregating a high proportion of asymptomatic I.P. has received grants from Duchenne Parent Project and benign cases responsible for founder effects. However, España (DPPE) and has received speaker honoraria it is also causative of significant functional impairment, from Genzyme Therapeutics and PTC Therapeutics. J. severe cardiac complications, cognitive alterations, and D-M. is an external advisor for Sanofi-Genzyme Spain potential shortening of life expectancy, the and has participated in advisory meetings for Sanofi-Gen- mechanopathogenesis of which remains largely unrevealed zyme, Amicus, Audentes, Sarepta, and Lupin. He has by our extensive search for modifying factors. Solving received funding for research from Sanofi-Genzyme, Ami- these issues would consolidate this mutation as one of the cus, Spark Therapeutics, and Boehringer-Ingelheim. He best potential models for DMD gene therapy, comparable has also received grants from DPPE, Fundacion Isabel to monoexon skipping therapies, with the advantage of Gemio, ISCIII, CIBERER, and AFM. The other authors being applicable to a broader spectrum of mutations. It declare no conflicts of interest. also preserves dystrophin functional domains, which is an issue of the minidystrophin gene transfer approach.20 To Data Availability achieve this goal, it is essential to overcome the hurdles The data that support the findings of this study are avail- derived from the usage of MES AO-cocktails or to the able from the corresponding author upon request. development of gene-editing therapies. Acknowledgments References 1. Darras BT, Urion DK, Ghosh PS. Dystrophinopathies. GeneReviews® We acknowledge Fundacion Isabel Gemio for the financial Last Update: January 20, 2022 [Internet]. University of Washington, support of this project and for the PhD grant to J.P.-G. Seattle; 1993-2022. 804 Volume 92, No. 5 Poyatos-García et al: DMD Δ45–55: Clinical Profile and Modifying Factors 2. North KN, Miller G, Iannaccone ST, et al. Cognitive dysfunction as 20. Happi Mbakam C, Lamothe G, Tremblay JP. 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Long-term efficacy of systemic tion of exons 45–55 of the Duchenne muscular dystrophy (DMD) multiexon skipping targeting dystrophin exons 45–55 with a cocktail gene. J Hum Genet 2009;54:127–130. of vivo-morpholinos in mdx52 mice. Mol Ther Nucleic Acids 2015;4: e225. 33. Geng C, Tong Y, Zhang S, et al. Sequence and structure characteris- tics of 22 deletion breakpoints in intron 44 of the DMD gene based 15. Lee J, Echigoya Y, Duddy W, et al. Antisense PMO cocktails effec- on long-read sequencing. Front Genet 2021;12:638220. tively skip dystrophin exons 45–55 in myotubes transdifferentiated from DMD patient fibroblasts. PLoS One 2018;13:e0197084. 34. Kaspar RW, Allen HD, Ray WC, et al. Analysis of dystrophin deletion mutations predicts age of cardiomyopathy onset in Becker muscular 16. Aoki Y, Yokota T, Nagata T, et al. Bodywide skipping of exons 45–55 dystrophy. Circ Cardiovasc Genet 2009;2:544–551. in dystrophic mdx52 mice by systemic antisense delivery. 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DMD exon 1 truncating point factor 6 mutation associated with myopathy and severe course of mutations: amelioration of phenotype by alternative translation initia- Becker muscular dystrophy. Neuromuscul Disord 2000;10: tion in exon 6. Hum Mutat 2009;30:633–640. 572–577. 806 Volume 92, No. 5